BackgroundThere is current interest in understanding the molecular mechanisms of tumor-induced bone pain. Accumulated evidence shows that endogenous formaldehyde concentrations are elevated in the blood or urine of patients with breast, prostate or bladder cancer. These cancers are frequently associated with cancer pain especially after bone metastasis. It is well known that transient receptor potential vanilloid receptor 1 (TRPV1) participates in cancer pain. The present study aims to demonstrate that the tumor tissue-derived endogenous formaldehyde induces bone cancer pain via TRPV1 activation under tumor acidic environment.Methodology/Principal FindingsEndogenous formaldehyde concentration increased significantly in the cultured breast cancer cell lines in vitro, in the bone marrow of breast MRMT-1 bone cancer pain model in rats and in tissues from breast cancer and lung cancer patients in vivo. Low concentrations (1∼5 mM) of formaldehyde induced pain responses in rat via TRPV1 and this pain response could be significantly enhanced by pH 6.0 (mimicking the acidic tumor microenvironment). Formaldehyde at low concentrations (1 mM to 100 mM) induced a concentration-dependent increase of [Ca2+]i in the freshly isolated rat dorsal root ganglion neurons and TRPV1-transfected CHO cells. Furthermore, electrophysiological experiments showed that low concentration formaldehyde-elicited TRPV1 currents could be significantly potentiated by low pH (6.0). TRPV1 antagonists and formaldehyde scavengers attenuated bone cancer pain responses.Conclusions/SignificanceOur data suggest that cancer tissues directly secrete endogenous formaldehyde, and this formaldehyde at low concentration induces metastatic bone cancer pain through TRPV1 activation especially under tumor acidic environment.

Oxalate secretion by fungi is known to be associated with fungal pathogenesis. In addition, oxalate toxicity is a concern for the commercial application of fungi in the food and drug industries. Although oxalate is generated through several different biochemical pathways, oxaloacetate acetylhydrolase (OAH)-catalyzed hydrolytic cleavage of oxaloacetate appears to be an especially important route. Below, we report the cloning of the Botrytis cinerea oahA gene and the demonstration that the disruption of this gene results in the loss of oxalate formation. In addition, through complementation we have shown that the intact B. cinerea oahA gene restores oxalate production in an Aspergillus niger mutant strain, lacking a functional oahA gene. These observations clearly indicate that oxalate production in A. niger and B. cinerea is solely dependent on the hydrolytic cleavage of oxaloacetate catalyzed by OAH. In addition, the B. cinera oahA gene was overexpressed in Escherichia coli and the purified OAH was used to define catalytic efficiency, substrate specificity, and metal ion activation. These results are reported along with the discovery of the mechanism-based, tight binding OAH inhibitor 3,3-difluorooxaloacetate (K i ‫؍‬ 68 nM). Finally, we propose that cellular uptake of this inhibitor could reduce oxalate production.Numerous filamentous fungi, including the food biotechnology fungus Aspergillus niger, the opportunistic human pathogen Aspergillus fumigatus, the phytopathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum, as well as many brown-rot and white-rot basidiomycetes, are able to efficiently produce large quantities of oxalate (1, 2). It is known that oxalate secretion is associated with fungal pathogenesis (1, 3-6). In the wood-rotting fungus Fomitopsis palustris oxalate is formed as the product of glucose metabolism (7). We recently initiated investigations of the oxalate biosynthetic pathway to develop a genomic-based method for distinguishing between oxalate producing and non-producing fungi. An additional goal of this effort was to identify enzyme inhibitors that could be used to arrest oxalate formation in targeted fungi.To attenuate oxalate production in fungi, it is necessary to first identify the major pathway responsible for oxalate formation. There are three potential routes for production of oxalate in fungi: oxidation of glyoxylate (8, 9), oxidation of glycolaldehyde (10), and hydrolysis of oxaloacetate (11). The results of studies of [ 14 C]CO 2 incorporation into the metabolite pools of A. niger indicate that oxalate is derived from oxaloacetate (12). This finding parallels the results of earlier work on the purification of an enzyme "oxalacetalase" (now known as oxaloacetate acetylhydrolase or OAH) 4 that catalyzes the hydrolytic cleavage of oxaloacetate to form acetate and oxalate (11). In a subsequent study, a mutant A. niger strain, NW228 (13), was found to be deficient in both oxalate production and in the synthesis of active OAH (14). These observations suggest that oxalate is ...

MicroRNAs (MiRNAs) have been found to be dysregulated in lung cancer tissues compared to their matched paracancerous tissues. However, the roles of miRNAs in peripheral blood as potential biomarkers for early diagnosis of lung cancer remain poorly understood. Here we found that miR-33a-5p and miR-128-3p were down-regulated in lung cancer tissues and cell lines. The expression levels of miR-33a-5p and miR-128-3p in lung cancer tissues were significantly correlated to TNM stages. MiR-128-3p in lung cancer tissues was also remarkably related to smoking and tumor size. The relative expression levels of miR-33a-5p and miR-128-3p were positively correlated in lung cancer tissues. Notably, miR-33a-5p and miR-128-3p in whole blood of lung cancer patients or early-stage lung cancer patients (TNM stage I-II) were lowly expressed as compared with that in healthy controls. The receiver operating characteristic curve (ROC) analyses revealed higher area under the ROC curve (AUC) values and higher sensitivity/specificity of miR-33a-5p and miR-128-3p alone and in combination were superior to that of traditional tumor markers (CYFR21-1, NSE and CA72-4). Importantly, both miR-33a-5p and miR-128-3p in whole blood were highly stable even under different harsh conditions. The results demonstrate that tumor suppressor miR-33a-5p/miR-128-3p in whole blood can serve as novel biomarkers for the early detection of lung cancer.

The work described in this paper was carried out to define the chemical function a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. This protein (Swiss-Prot entry Q05957) is synthesized in the senescent flower petals and is named the "petal death protein" or "PDP". On the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily, a substrate screen that employed a (2R)-malate core structure was designed. Accordingly, stereochemically defined C(2)- and C(3)-substituted malates were synthesized and tested as substrates for PDP-catalyzed cleavage of the C(2)-C(3) bond. The screen identified (2R)-ethyl, (3S)-methylmalate, and oxaloacetate [likely to bind as the hydrate, C(2)(OH)(2) gem-diol] as the most active substrates (for each, k(cat)/K(m) = 2 x 10(4) M(-)(1) s(-)(1)). In contrast to the stringent substrate specificities previously observed for the Escherichia coli isocitrate and 2-methylisocitrate lyases, the PDP tolerated hydrogen, methyl, and to a much lesser extent acetate substituents at the C(3) position (S configuration only) and hydoxyl, methyl, ethyl, propyl, and to a much lesser extent isobutyl substituents at C(2) (R configuration only). It is hypothesized that PDP functions in oxalate production in Ca(2+) sequestering and/or in carbon scavenging from alpha-hydroxycarboxylate catabolites during the biochemical transition accompanying petal senescence.

Our results provide novel evidence for the increase of IGF-1 in tibia bone marrow, which is responsible for the up-regulation of TRPV1 expression and function in the peripheral nerves of bone cancer pain rats.

Previous work has indicated that PEP mutase catalyzes the rearrangement of phosphoenolpyruvate to phosphonopyruvate by a dissociative mechanism. The crystal structure of the mutase with Mg(II) and sulfopyruvate (a phosphonopyruvate analogue) bound showed that the substrate is anchored to the active site by the Mg(II), and shielded from solvent by a large loop (residues 115-133). Here, the crystal structures of wild-type and D58A mutases, in the apo state and in complex with Mg(II), are reported. In both unbound and Mg(II)-bound states, the active site is accessible to the solvent. The loop (residues 115-133), which in the enzyme-inhibitor complexes covers the active site cavity, is partially disordered or adopts a conformation that allows access to the cavity. In the apo state, the residues associated with Mg(II) binding are poised to accept the metal ion. When Mg(II) binds, the coordination is the same as that previously observed in the enzyme-Mg(II) sulfopyruvate complex, except that the coordination positions occupied by two ligand oxygen atoms are occupied by two water molecules. When the loop opens, three key active site residues are displaced from the active site, Lys120, Asn122, and Leu124. Lys120 mediates Mg(II) coordination. Asn122 and Leu124 surround the transferring phosphoryl group, and thus prevent substrate hydrolysis. Amino acid replacement of any one of these three loop residues results in a significant loss of catalytic activity. It is hypothesized that the loop serves to gate the mutase active site, interconverting between an open conformation that allows substrate binding and product release and a closed conformation that separates the reaction site from the solvent during catalysis.Conversion of phosphoenolpyruvate (PEP) 1 to phosphonopyruvate (P-pyr) is catalyzed by PEP mutase, a 38 kDa tetrameric Mg(II)-dependent enzyme (1, 2):This P-C bond forming reaction serves as the point of entry to all known phosphonate biosynthetic pathways (3). The crystal structure of the PEP mutase from the mollusk Mytilus edulis was determined in the presence of Mg(II) oxalate, a tight binding Mg(II) pyruvate enolate homologue (4), revealing a tetrameric structure in which each molecule adopts an R/ barrel fold, with pairwise swapping of the eighth helix. To further investigate the catalytic mechanism, a second structure of the enzyme was determined, that in complex with the inhibitor sulfopyruvate (S-pyr), a compound isosteric with P-pyr with the sulfur replacing phosphorus. That structure, in conjunction with a site-directed mutagenesis probe of potential catalytic residues, suggested that catalysis proceeds via a dissociative mechanism involving the intermediacy of metaphosphate and the enolate anion of pyruvate (5). Both Mg(II) oxalate and Mg(II) S-pyr bind in the same location, at the center of the barrel, close to the C-terminal ends of the -strands, and in both cases, two loop regions and part of the kinked C-terminal helix of the neighboring molecule bury the inhibitors and prevent access to th...

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range. The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg2+ binding first (Kd = 140 +/- 40 microM), are kcat = 105 +/- 2 s(-1) and P-pyr Km = 5 +/- 1 microM. PEP (slow substrate kcat = 2 x 10(-4) s(-1)), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 +/- 0.1 mM, 17 +/- 1 microM, and 210 +/- 10 microM, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (alpha/beta)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.

circRNA CDR1as (CDR1as) has been demonstrated to play important roles in a variety of inflammation-related diseases by acting as miRNA sponges. The present study is aimed at investigating the potential roles of CDR1as in the proliferation of human periodontal ligament stem cells (PDLSCs) under an inflammatory condition induced by Porphyromonas gingivalis-derived lipopolysaccharide (LPS). Human periodontal ligament cells (PDLCs) were isolated from periodontal ligament tissue, and PDLSCs were sorted from PDLCs based on the STRO-1 expression through fluorescence-activated cell sorting. We further found that CDR1as was significantly downregulated in LPS-treated PDLSCs compared to untreated cells, as well as in normal periodontal ligament tissues compared to periodontitis tissues. Knockdown of CDR1as promoted LPS-induced proliferative inhibition of PDLSCs, whereas overexpression of CDR1as alleviated the LPS-induced proliferative ability of PDLSCs. Mechanistically, CDR1as functioned as an miR-7 sponge to activate the ERK signal pathway to mediate the inhibition effect of LPS on cell proliferation. Taken together, our findings revealed the effects of the interacting pair of CDR1as/miR-7 on the proliferation ability of PDLSCs within their surrounding inflammatory microenvironment of periodontitis.